Epithelial barrier function is regulated to a large extent by the apical most intercellular junction referred to as the tight junction (TJ). The TJ not only separates the lumenal compartment from the tissue space, but also regulates movement of solutes across the paracellular space in diverse physiologic and pathologic states. Defects in epithelial barrier and changes in TJs have been observed in mucosal specimens from individuals with inflammatory bowel disease at both a functional and structural level. We recently cloned the human homolog of a novel TJ-associated membrane protein termed junction adhesion molecule (JAM). Our preliminary data suggests an important role of this protein in regulating paracellular permeability and TJ organization in intestinal epithelia. Thus, the overall objective of this proposal is to define on a structural and functional level the mechanism(s) by which JAM regulates TJ assembly and epithelial barrier function. In this proposal, we will test two hypotheses: Our first hypothesis is that the extracellular domain of JAM is critical for protein function and mediates the formation of intercellular junctions. This hypothesis will be tested by examining the effects of a panel of JAM extracellular domain-specific mAbs on barrier function and TJ reassembly followed by mapping the epitopes for these mAbs. In addition, the effect of recombinant JAM and mutant JAM constructs on cell-cell interactions will be examined. Our second hypothesis is that the cytoplasmic tail of JAM is necessary for specific interactions with TJ protein family members and signal transduction proteins that regulate paracellular permeability. We will test this hypothesis by extending preliminary studies on TJ associated proteins that bind to regions of the cytoplasmic tail of JAM and by analysis of cell-cell junctions formed by mutant JAM constructs. Information from these studies should provide mechanistic insights into the regulation of TJ function and potential new therapeutic avenues aimed at modulating mucosal permeability.